The productive phase of the HPV life cycle is restricted to the uppermost layer of the epithelium, in cells that have normally exited the cell cycle. The E7 protein alters cell cycle regulation to push differentiating cells back into the cell cycle, allowing for productive replication and virion production in a G2-arrested environment. Unscheduled cell cycle re-entry, while necessary for viral replication, results in replication stress that can lead to genomic instability, contributing to cancer development. E7 is able to bypass senescence, a stable growth arrest associated with replication stress, by targeting the Rb tumor suppressor for degradation. Our long-term goal is to identify signaling pathways that regulate viral replication in order to understand the mechanisms by which HPV persists and causes cancer. This proposal focuses on understanding how HPV hijacks the ATR DNA damage pathway to facilitate viral replication. We will identify the mechanisms by which HPV utilizes ATR and its downstream target Chk1 to increase the levels of RRM2, a key component in nucleotide synthesis that is necessary for viral replication. We will also determine if ATR activity is required for survival of HPV-infected cells in response to E7-induced replication stress. We hypothesize that high-risk HPV positive cells are addicted to the replication stress-protective role ATR/Chk1/RRM2. Specific Aims to test this are: (1) To determine how HPV increases the levels of RRM2 through E7 by identifying domains in E7 necessary for increased RRM2 protein stability. We will also determine the contribution of ATR activity to the increased stability of RRM2 protein and elevated dNTP pools in HPV positive cells. (2) To determine if ATR activity protects HPV infected cells from E7- induced replication stress by using ATR, Chk1 and RRM2 inhibitors and small hairpin RNAs (shRNAs) and examining markers of replication stress and DNA damage on cellular, as well as viral DNA. We will also determine if the ATR-mediated increase in RRM2 is necessary for HPV to bypass oncogene-induced senescence (OIS) during the process of cellular immortalization, and to protect established HPV-infected lines from OIS by examining the effect of ATR and RRM2 depletion on cell growth arrest, senescence markers, and apoptosis. ATR is the central regulator of genome stability in response to replication stress. Understanding how ATR activity promotes the viral life cycle will provide insight into mechanisms of viral persistence, as well as genomic instability. These studies may also identify therapeutic targets for the treatment of HPV-associated diseases.